The development of fibrosis represents a critical parameter of airway remodeling, and likely is the outcome of numerous signaling cascades operative in different cell types. The pro-fibrotic cytokine, transforming growth factor beta-1 (TGF-1) plays a cardinal role in the development of fibrosis, and the outcome of TGF-1 induced signaling is highly dependent upon the cellular environment, and the cooperation with other signaling pathways. Exciting new preliminary studies from our laboratory have identified a critical role for c-Jun-N- terminal-kinase (JNK) in augmenting the pro-fibrotic effects of TGF-1, in association with the causation of a mesenchymal transition of airway epithelial cells (EMT). We have demonstrated that; 1) JNK is predominantly activated in airway epithelium from ovalbumin sensitized and challenged mice; 2) Mice lacking JNK1 (JNK1-/-) fail to induce mesenchymal genes or develop fibrosis in response to ovalbumin challenge or TGF-1, or bleomycin administration; 3) Primary tracheal epithelial cells derived from JNK1-/- mice are refractory to TGF-1-induced expression of mesenchymal genes and proteins; and 4) TGF-1 induced epithelial-to mesenchymal transition requires JNK1. Our data, suggest a critical role of airway epithelium-derived JNK- dependent signals in orchestrating airways fibrosis. The hypothesis of this proposal is that activation of JNK1 in the airway epithelium is required for the development of subepithelial fibrosis in murine models of airway remodeling by promoting epithelial to mesenchymal transition (EMT). We hypothesize that JNK1 enhances TGF-1 signaling, via coordinated linker phosphorylation of receptor SMADs, and phosphorylation of c-Jun, resulting in enhanced expression of mesenchymal genes, critical to TGF-1-induced fibrosis. In Specific Aim #1, we will determine the requirement for JNK1 in TGF-1-induced epithelial to mesenchymal transition in primary cultures of tracheal epithelial cells. In Specific Aim #2, we will determine whether increases in mesenchymal gene expression in airway epithelium, and development of subepithelial fibrosis in mice require a JNK1-TGF- signaling axis. Specific Aim #3 proposes to elucidate the relative importance of JNK1- dependent phosphorylation of c-Jun or rSMADs linker regions in augmenting TGF-1 induced signaling, whereas Specific Aim #4 will unravel whether JNK1 activation, c-Jun and SMAD3 linker phosphorylation, and subsequent c-Jun and SMAD4 dependent transcriptional responses within the airway epithelium are critical in the orchestration of OVA or TGF-1-induced airways fibrosis. The outcome of our study, which is based on cell culture and genetically altered mouse models, will provide a better understanding of the molecular signals that lead to collagen deposition (scarring) of airways, and the relevance of epithelial cells in this process. Project Narrative: Epithelial cells that line the airways (breathing tubes) are now well known to play a critical role in the defense against inhaled materials, and are important in maintaining normal lung function. However, our laboratories have identified that epithelial cells play a negative role in promoting stiffening of the lung tissue, thereby decreasing lung function. We have identified some critical biochemical signals that promote this possibly negative event. Project Narrative: This grant proposal has four specific aims to test the importance of these biochemical signals in lung stiffening, and will use primary cell cultures from mouse airways, a human epithelial cell line, and the construction of genetically altered mice. Completion of this project will provide new insights into the process of lung stiffening, and may lead to the development of new therapeutics to limit lung stiffening, and also potentially reverse this process. [unreadable] [unreadable] [unreadable]